In vitro nucleic acid amplification techniques have provided powerful tools for detection and analysis of small amounts of nucleic acids. The extreme sensitivity of such methods has lead to attempts to develop them for diagnosis of infectious and genetic diseases, isolation of genes for analysis, and detection of specific nucleic adds as in forensic medicine. Nucleic acid amplification techniques can be grouped according to the temperature requirements of the procedure. The polymerase chain reaction (PCR; R. K. Saiki, et al. 1985, Science 230, 1350-1354), ligase chain reaction (LCR; D. Y. Wu, et al. 1989, Genomics 4, 560-569; K. Barfinger, et al. 1990. Gene 89, 117-122; F. Barany. 1991. Proc. Natl. Acad. Sci. USA 88, 189-193) and transcription-based amplification (D. Y. Kwoh, et al. 1989. Proc. Natl. Acad. Sci. USA 86, 1173-1177) require temperature cycling. In contrast, methods such as strand displacement amplification (SDA; G. T. Walker, et al. 1992. Proc. Natl. Acad Sci. USA 89, 392-396 and G. T. Walker, et al. 1992. Nuc. Acids. Res. 20, 1691-1696, both disclosures being incorporated herein by reference), self-sustained sequence replication (3SR; J. C. Guatelli, et al. 1990. Proc. Natl. Acad. Sci. USA 87, 1874-1878) and the Qβ replicase system (P. M. Lizardi, et al. 1988. BioTechnology 6, 1197-1202) are isothermal reactions. In addition, WO 90/10064 and WO 91/03573 describe use of the bacteriophage phi29 replication origin for isothermal replication of nucleic acids.
A variety of methods have also been developed to detect and/or measure nucleic acid amplification. For the most part, these methods are primer-based, meaning that they depend on hybridization of a primer to the target sequence, in some cases followed by extension of the primer. Primer-based detection of amplified nucleic acids in PCR often relies on incorporation of an amplification primer into the amplified product (amplicon) during the amplification reaction. Features engineered into the PCR amplification primer therefore appear in the amplification product and can be used either to detect the amplified target sequence or to immobilize the amplicon for detection by other means. For example, Syvanen, et al. (1988. Nucleic Acids Res. 16, 11327-11338) report the use of biotinylated PCR amplification primers to produce biotin-containing amplification products. These amplicons can then be hybridized to a second probe containing a fluorescent dye or other reporter group. The hybridized complex is then selectively isolated from other components of the reaction mixture by affinity-based immobilization of the biotin-containing complex and is detected by means of the reporter group. Laongiaru, et al. (1991. European Patent Application No. 0 420 260) describe a similar use of biotin-containing PCR amplification primers conjugated to fluorescent dyes for detection of PCR amplification products. The amplicons containing the primers are separated from unextended primers on the basis of size, and multiplex amplification was detected using different fluorescent dyes on two amplification primer sets. Kemp, et al. (1989. Proc. Natl. Acad. Sci. USA 86, 2423-2427; 1990. PCT Patent Application No. WO 90/06374) describe a method for capturing amplified DNA by incorporation of one modified amplification primer and use of a second modified amplification primer as a means for detection. The Kemp “capture primer[ contains a 5′ tail which is the single stranded form of the recognition sequence for the double-stranded DNA binding protein GCN4. The Kemp “detector primer” includes a biotin moiety on its 5′ end. The amplified product is immobilized by binding to the double-stranded GCN4 recognition sequence generated by amplification using the capture primer. The biotin moiety introduced by the detector primer is bound to an avidin-peroxidase complex to provide colorimetric detection of the immobilized PCR amplification product. Wahlberg, et al. (1990. Proc. Natl. Acad. Sci. USA 87, 6569-6573) report a similar method in which one PCR amplification primer is biotinylated and the other contains a 5′ tail encoding the E. coli lac operator sequence. Double stranded amplification products are immobilized by binding to streptavidin and detected colorimetrically by binding of a lac repressor-β-galactosidase fusion protein to the double-stranded lac operator generated by amplification. The Wahlberg, et al. method differs from the Kemp, et al. method in that the biotin-streptavidin interaction rather than the double-stranded binding protein provides immobilization of the amplification products and the double-stranded binding protein provides colorimetric detection. This suggests that the two methods could be combined by using two amplification primers, each with a 5′ tail encoding the recognition sequence of a different double-stranded binding protein. Amplified products could then be immobilized by binding to one double stranded binding protein and detected by binding to the other. C. A. Vary (1992. Clinical Chemistry 38, 687-694; 1992. PCT Patent Application No. WO 92/11390) describes the use of amplification primers containing 5′ tails which form hybridization sites for a third oligonucleotide when incorporated into otherwise double-stranded amplicons. Hybridization of one tail was used to capture the amplified product and the other was used to detect it by hybridization to a probe conjugated to a fluorescent dye.
All of these primer-based methods of detecting PCR amplification products require two amplification reactions to achieve high sensitivity, i.e., detection of fewer than 100 copies of the target sequence. That is, a first amplification of the target sequence is followed by a second amplification using nested primers incorporating the desired modifications for capture and/or detection. Two consecutive amplifications in this manner are needed to avoid unacceptably high levels of background signal produced by amplification of non-target DNA spuriously primed with the modified, signal-generating primers. This feature of the prior art methods makes them time-consuming and cumbersome, and the advantages of primer-based detection methods are therefore often offset by the requirement for a second consecutive amplification reaction.
Non-specific amplification of DNA would be expected to present particular problems for primer-based detection of amplification products in SDA reactions because these amplifications are carried out at a relatively low temperature (about 37°-40° C.) which would allow increased mispriming as compared to PCR, resulting in even higher levels of background signal. Unexpectedly, the instant methods for primer-based detection of SDA resulting in low levels of background signal in spite of the use of only a single amplification reaction which generates products for detection concurrently with amplification of the target sequence. Simultaneous or concurrent generation of a secondary amplification product and the amplified target sequence is referred to herein as real-time primer extension, real-time detection of amplification, etc.